MCL-1 siRNA通过线粒体凋亡途径部分逆转耐顺铂结肠癌细胞系SW480的耐药性

目的 探讨顺铂耐药结肠癌细胞的MCL-1表达水平与顺铂耐药性的关系。方法 用MTT法检测顺铂耐药结肠癌细胞系SW480(SW480-R)对顺铂的敏感性。通过检测siRNA下调SW480-R 细胞MCL-1的表达作用观察其对细胞耐药性的影响。Western blot试验检测SW480-R 细胞Bcl-2家族蛋白及线粒体来源促凋亡因子的表达水平。Annexin V/PI染色检测SW480-R 细胞的凋亡。结果 SW480-R细胞相比于常规SW480细胞对顺铂的敏感性显著下降,western blot结果表明SW480-R细胞的MCL-1水平显著上调而其他Bcl-2蛋白家族成员(Bcl-2,Bcl-xl,BIM,BAK,BAX)表达水平变化不明显。体外转染MCL-1 siRNA能逆转SW480-R细胞的耐药性,提高顺铂对SW480-R的杀伤活性。在SW480-R细胞中,MCL-1 siRNA能促进线粒体来源的促凋亡因子(细胞色素C、凋亡诱导因子、Smac/DIABLO)在顺铂治疗下从线粒体中释放到细胞质中,进而诱导耐药肿瘤细胞发生凋亡。结论 MCL-1的高表达可能是结肠癌细胞产生顺铂耐药性的重要机制,MCL-1基因沉默能通过线粒体凋亡途径逆转耐顺铂结肠癌细胞系SW480的耐药性。     

MiR-19反义核酸与CD133+HT29细胞亚群对多柔比星敏感性关系的研究

目的 探讨miR-19反义核酸与CD133⁺HT29细胞亚群对多柔比星敏感性的关系并研究其机制。方法 用RT-qPCR方法检测miR-19在结直肠癌细胞中的表达水平。流式细胞术检测miR-19反义核酸和多柔比星对HT29细胞系的CD133⁺细胞亚群种群比例的影响。MTT法检测miR-19反义核酸对多柔比星杀伤CD133⁺HT29细胞亚群能力的影响。利用生物信息学及Western blot法验证miR-19是否调节CD133⁺HT29细胞亚群中PTEN的表达。运用Western blot、免疫共沉淀、流式细胞术研究miR-19反义核酸影响多柔比星疗效的信号通路。结果 结直肠癌细胞系的miR-19表达水平显著高于正常结直肠上皮细胞系,并且CD133⁺细胞中的miR-19表达水平显著高于常规结直肠癌细胞。多柔比星体外单独治疗能提高HT29细胞系中CD133⁺细胞亚群的比例,然而联用miR-19反义核酸后CD133⁺HT29细胞亚群的种群比例显著下降。MTT结果表明miR-19反义核酸可显著增强多柔比星对CD133⁺HT29细胞亚群的杀伤活性。Western blot实验表明miR-19的靶基因可能为PTEN。MiR-19反义核酸可显著抑制CD133⁺HT29细胞亚群PI3K、AKT和Bad的磷酸化,增强Bad与Bcl-2和Bcl-xl的结合,从而提高CD133⁺HT29细胞亚群对多柔比星依赖的凋亡信号的敏感性,促进caspase-9和caspase-3的活化。结论 MiR-19反义核酸通过PTEN/PI3K/AKT/Bad途径提高CD133⁺HT29细胞亚群对多柔比星的敏感性。     

miR-346靶向调控SFRP4介导5-FU诱导的结肠癌细胞毒性

目的观察miR-346及SFRP4对5-FU诱导的结肠癌细胞毒性的影响,为结肠癌化疗增效提供新的靶基因。方法构建pcDNA3.1-SFRP4上调SFRP4表达,以pcDNA3.1-SFRP4、NC、Con-miR、miR-346、miR-346+Vector、miR-346+SFRP4等转染SW480和HT-29细胞,Western blot法观察miR-346上调或下调后对SFRP4蛋白水平的影响,MTT观察不同转染条件下5-FU细胞存活率变化,流式细胞术观察细胞凋亡变化。结果 SFRP4可增加5-FU诱导的SW480、HT-29细胞毒性及细胞凋亡,miR-346能够降低5-FU诱导的SW480、HT-29细胞毒性及细胞凋亡。miR-346对SFRP4表达具有负向调控作用。结论 miR-346通过负向调控SFRP4表达降低5-FU诱导的结肠癌细胞毒性。     

MiR-182对乳腺癌细胞顺铂耐药的相关性研究

目的 探讨miR-182与乳腺癌细胞顺铂耐药性的关系。方法 MTT法检测miR-182对顺铂杀伤乳腺癌细胞能力的影响。利用生物信息学、定量PCR及western blot法验证miR-182是否能调节乳腺癌细胞BNIP3的表达。运用JC-1染色、Annexin V染色及western blot法研究miR-182影响顺铂疗效的信号通路。结果 miR-182模拟物可减弱顺铂对MCF-7细胞的杀伤活性,而miR-182抑制剂则增强顺铂对MCF-7细胞的杀伤活性。定量PCR及western blot实验表明miR-182的靶基因可能为BNIP3。miR-182抑制剂联合顺铂可引起MCF-7细胞线粒体膜电位显著下降并诱导caspase-3的活化和凋亡的发生,转染BNIP3 siRNA后miR-182抑制剂联合顺铂对MCF-7细胞的凋亡诱导效应显著降低。结论 MiR-182在乳腺癌中通过下调BNIP3的表达影响顺铂对乳腺癌细胞的杀伤活性。     

细胞粘附在结肠癌细胞系SW480多药耐药表型中的作用

目的探讨细胞粘附在结肠癌细胞系SW 480多药耐药表型中的作用。方法通过细胞粘附实验、四甲基偶氮唑盐实验（MTT法）、细胞内阿霉素的蓄积和潴留及Annexin V/PI染色法检测凋亡等检测粘附于层粘连蛋白（LN）的结肠癌细胞系SW 480的多药耐药性的改变。结果粘附于LN的SW 480细胞对化疗药物敏感性显著下降,化疗药物的IC50均显著升高,化疗药物诱导的凋亡指数明显减少 细胞内阿霉素的蓄积和潴留均明显减少,药物泵出率显著增加（P〈0.05）。结论结肠癌细胞SW 480与LN粘附后,可能提高了对药物转运的能力,导致化疗药物在肿瘤细胞内蓄积的减少 通过提高抗凋亡的能力,逃避化疗药物诱导的凋亡。     

雷公藤红素促进RIP1蛋白的去泛素化增强TNF-α对结肠癌细胞的凋亡诱导活性的研究

目的 探讨中药活性成分雷公藤红素对TNF-α体外抗结肠癌活性的影响并研究其机制。方法 用雷公藤红素联合TNF-α体外治疗结肠癌细胞系SW480,MTT法检测SW480细胞的细胞活力。SW480细胞用雷公藤红素联合TNF-α治疗后,Annexin V/PI染色法检测细胞的凋亡,Western blot法检测细胞caspase-8、caspase-9和caspase-3的活化和对CYLD蛋白表达的影响,并用免疫共沉淀法检测SW480细胞RIP1蛋白的泛素化水平。结果 雷公藤红素联合TNF-α对结肠癌细胞系SW480的细胞活力抑制率和凋亡诱导活性均显著高于雷公藤红素及TNF-α单治疗组。雷公藤红素联合TNF-α对SW480细胞caspase-8、caspase-9和caspase-3的活化显著高于雷公藤红素及TNF-α单治疗组,且两者联合治疗后,caspase-8的活化时间显著早于caspase-9和caspase-3。SW480细胞用雷公藤红素联合TNF-α治疗后,其RIP1蛋白的泛素化水平显著低于雷公藤红素及TNF-α单治疗组。进一步研究发现,雷公藤红素能显著诱导SW480细胞CYLD蛋白的表达,而TNF-α对CYLD的表达水平无影响,当用小干扰RNA沉默CYLD的表达后,雷公藤红素对TNF-α的协同效应丧失。结论 雷公藤红素通过促进RIP1蛋白的去泛素化增强TNF-α对结肠癌细胞的凋亡诱导活性。     

miR-27a对两种结肠癌细胞增殖和侵袭能力的影响

目的探讨microRNA-27a(miR-27a)对2种结肠癌细胞增殖和侵袭能力的影响。方法应用miR-27a反义寡核苷酸(antisense oligonucleotides,ASO)在体外转染结肠癌细胞系SW620和HCT116,设置转染无义序列组作为对照组。RT-PCR检测细胞miR-27a表达;MTT法检测细胞增殖;Transwell小室侵袭实验检测细胞侵袭能力;Western blot检测转染细胞FOXO1蛋白表达。结果与对照组相比,转染miR-27a ASO后结肠癌细胞系SW620和HCT116的miR-27a表达水平均明显降低(P<0.001),增殖能力和侵袭能力均受到明显抑制(P<0.01),FOXO1蛋白表达均明显增高(P<0.05)。结论抑制miR-27a表达可显著降低结肠癌细胞系SW620和HCT116的增殖能力和侵袭能力,而调控下游FOXO1蛋白表达可能是miR-27a参与结肠癌细胞增殖和侵袭的主要机制。     

槲皮素通过抑制己糖激酶的表达促进结肠癌SW480细胞凋亡

目的观察槲皮素对SW480细胞增殖和凋亡的影响并探讨其机制。方法采用流式细胞检测技术检测槲皮素诱导人结肠癌SW480细胞的凋亡能力;应用免疫细胞化学技术检测细胞中己糖激酶蛋白的表达。结果槲皮素以浓度依赖性的方式诱导结肠癌SW480细胞的凋亡并下调己糖激酶蛋白的表达。结论槲皮素通过抑制己糖激酶的表达来促进结肠癌SW480细胞凋亡。     

藏药湿生扁蕾提取物通过NF-κB通路诱导结肠癌细胞凋亡的机制研究

目的探讨藏药湿生扁蕾提取物通过NF-κB信号通路诱导结肠癌SW480细胞凋亡的作用及其机制。方法本研究用不同浓度的藏药湿生扁蕾提取物处理结肠癌SW480细胞,通过JC-1染色检测SW480细胞线粒体膜电位的变化,通过Hoechst染色以判断藏药湿生扁蕾提取物是否具有诱导结肠癌细胞SW480细胞凋亡效应。同时检测NF-κB蛋白的表达,用以明确NF-κB信号通路在结肠癌发生中的作用机制。结果经一定浓度的湿生扁蕾提取物处理后的SW480细胞内,NF-κB的表达明显下调,染色现象揭示湿生扁蕾提取物具有促进肿瘤细胞凋亡的作用。结论湿生扁蕾提取物可以在体外诱导肿瘤细胞凋亡,其抗肿瘤机制是通过NF-κB信号通路介导的。     

藏药湿生扁蕾提取物通过NF-κB通路诱导结肠癌细胞凋亡的机制研究

目的：探讨藏药湿生扁蕾提取物通过 NF －κB 信号通路诱导结肠癌 SW480细胞凋亡的作用及其机制。方法本研究用不同浓度的藏药湿生扁蕾提取物处理结肠癌 SW480细胞,通过 JC －1染色检测 SW480细胞线粒体膜电位的变化,通过 Hoechst 染色以判断藏药湿生扁蕾提取物是否具有诱导结肠癌细胞 SW480细胞凋亡效应。同时检测 NF －κB 蛋白的表达,用以明确 NF －κB 信号通路在结肠癌发生中的作用机制。结果经一定浓度的湿生扁蕾提取物处理后的 SW480细胞内,NF －κB 的表达明显下调,染色现象揭示湿生扁蕾提取物具有促进肿瘤细胞凋亡的作用。结论湿生扁蕾提取物可以在体外诱导肿瘤细胞凋亡,其抗肿瘤机制是通过 NF －κB 信号通路介导的。     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Aquatic Insects and Trace Metals: Bioavailability,Bioaccumulation, and Toxicity

Abstract

The uptake of metals from food and water sources by insects is thought to be additive. For a given metal, the proportions taken up from water and food will depend both on the bioavailable concentration of the metal associated with each source and the mechanism and rate by which the metal enters the insect. Attempts to correlate insect trace metal concentrations with the trophic level of insects should be made with a knowledge of the feeding relationships of the individual taxa concerned. Pathways for the uptake of essential metals, such as copper and zinc, exist at the cellular level, and other nonessential metals, such as cadmium, also appear to enter via these routes. Within cells, trace metals can be bound to proteins or stored in granules. The internal distribution of metals among body tissues is very heterogeneous, and distribution patterns tend to be both metal and taxon specific. Trace metals associated with insects can be both bound on the surface of their chitinous exoskeleton and incorporated into body tissues. The quantities of trace meals accumulated by an individual reflect the net balance between the rate of metal influx from both dissolved and particulate sources and the rate of metal efflux from the organism. The toxicity of metals has been demonstrated at all levels of biological organization: cell, tissue, individual, population, and community. Much of the literature pertaining to the toxic effects of metals on aquatic insects is based on laboratory observations and, as such, it is difficult to extrapolate the data to insects in nature. The few experimental studies in nature suggest that trace metal contaminants can affect both the distribution and the abundance of aquatic insects. Insects have a largely unexploited potential as biomonitors of metal contamination in nature. A better understanding of the physico-chemical and biological mechanisms mediating trace metal bioavailability and exchange will facilitate the development of general predictive models relating trace metal concentrations in insects to those in their environment. Such models will facilitate the use of insects as contaminant biomonitors.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.     

Alzheimer’s disease – current trends in basic and clinical research. Highlights from the 16th ECNP

Advances in the molecular biological knowledge of neuronal nicotinic acetylcholine receptors (nAChRs) have led to a growing interest by the pharmaceutical industry in the development of novel compounds that selectively modulate nAChR function. The ability of (-)-nicotine, an activator of nAChRs, to enhance attentional aspects of cognition in animals and humans, to exert neuroprotective and anxiolytic-like effects, and presumably to mediate the negative correlation between smoking and Alzheimer's (and Parkinson's) Disease, has focused interest on the potential therapeutic utility of modulators of nAChR function for treatment of some of the deficits associated with these progressive, neurodegenerative conditions. Numerous compounds are known which activate nAChRs and which might serve as lead compounds toward the development of such agents. The pharmacologic diversity of neuronal nAChR subtypes suggests the possibility of developing selective compounds which would have more favourable side-effect profiles than existing agents. This broader class of agents, collectively called cholinergic channel modulators (ChCMs), is anticipated to encompass compounds which would have more favourable side-effect profiles than existing agents, which generally exhibit low selectivity. This selectivity may be achieved by preferentially activating some subtypes of nAChRs (i.e., Cholinergic Channel Activators, ChCAs) or inhibiting the function of other subtypes (Cholinergic Channel Inhibitors, ChCIs). An overview of the biology of nAChRs and the rationale for the use of ChCMs for the treatment of dementia related to neurodegenerative diseases are presented, followed by a discussion of lead compounds and compounds under consideration for clinical evaluation.